Structural genomics is the largest contributor of novel structural leverage

The Protein Structural Initiative (PSI) at the US National Institutes of Health (NIH) is funding four large-scale centers for structural genomics (SG). These centers systematically target many large families without structural coverage, as well as very large families with inadequate structural coverage. Here, we report a few simple metrics that demonstrate how successfully these efforts optimize structural coverage: while the PSI-2 (2005-now) contributed more than 8% of all structures deposited into the PDB, it contributed over 20% of all novel structures (i.e. structures for protein sequences with no structural representative in the PDB on the date of deposition). The structural coverage of the protein universe represented by today’s UniProt (v12.8) has increased linearly from 1992 to 2008; structural genomics has contributed significantly to the maintenance of this growth rate. Success in increasing novel leverage (defined in Liu et al. in Nat Biotechnol 25:849–851, 2007) has resulted from systematic targeting of large families. PSI’s per structure contribution to novel leverage was over 4-fold higher than that for non-PSI structural biology efforts during the past 8 years. If the success of the PSI continues, it may just take another ~15 years to cover most sequences in the current UniProt database.     

Pentapeptide repeat proteins

The pentapeptide repeat protein (PRP) family has more than 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S,T,A,V][D,N][L,F][S,T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral beta-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable similarity in size, shape, and electrostatics to DNA.     

Studies on solvatochromic properties of aminophenylstyryl-quinolinum dye, LDS 798, and its application in studying submicron lipid based structure

The styryl group of dyes has been used in cellular studies for over 20 years because of their solvatochromic and/or electrochromic properties. Here we report characterization of solubility and solvatochromic properties of a near infra-red styryl dye, styryl 11 or LDS 798. We have extended our studies to small unilamellar vesicles and lipid based nanoparticles and found that solvatochromic properties of this dye used in tandem with fluorescence correlation spectroscopy can be used to efficiently determine the diffusion coefficient and hence the size of the submicron lipid based particles. This technique has the potential to provide essential information about liposomal and vesicular structures and their movement in vitro and in situ.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N backbone chemical shift assignments of 4E-BP1<Subscript>44–87</Subscript> and 4E-BP1<Subscript>44–87</Subscript> bound to eIF4E

The eukaryotic translational initiation factor 4G (eIF4G) interacts with the cap-binding protein eIF4E through a consensus binding motif, Y(X)₄LΦ (where X is any amino acid and Φ is a hydrophobic residue). 4E binding proteins (4E-BPs), which also contain a Y(X)₄LΦ motif, regulate the eIF4E/eIF4G interaction. The non- or minimally-phosphorylated form of 4E-BP1 binds eIF4E, preventing eIF4E from interacting with eIF4G, thus inhibiting translation initiation. 4EGI-1, a small molecule inhibitor of the eIF4E/eIF4G interaction that is under investigation as a novel anti-cancer drug, has a dual activity; it disrupts the eIF4E/eIF4G interaction and stabilizes the binding of 4E-BP1 to eIF4E. Here, we report the complete backbone NMR resonance assignment of an unliganded 4E-BP1 fragment (4E-BP1_44–87). We also report the near complete backbone assignment of the same fragment in complex to eIF4E/m⁷GTP (excluding the assignment of the last C-terminus residue, D87). The chemical shift data constitute a prerequisite to understanding the mechanism of action of translation initiation inhibitors, including 4EGI-1, that modulate the eIF4E/4E-BP1 interaction.     

Immobilized Triton X-100-assisted refolding of Green Fluorescent Protein-Tobacco Etch Virus protease fusion protein using β-cyclodextrin as the eluent

A new protein refolding technique based on the use of the non-charged detergent Triton X-100 immobilized to the cross-linked agarose gel Sepharose High Performance has been developed. The new solid phase was used in combination with soluble β-cyclodextrin (β-CD) to refold recombinant Green Fluorescent Protein fused to Tobacco Etch Virus protease (GFPTEVP) expressed as inclusion bodies in E. coli. Previous attempts to refold recombinant GFPTEVP by dilution had failed. In the new procedure a column packed with Triton X-100-coupled Sepharose High Performance was used to capture unfolded GFPTEVP followed by elution using an increasing β-CD concentration gradient. The yield of properly refolded GFPTEVP was 46% at a protein concentration of 380 μg/ml. In contrast, dilution refolding of GFPTEVP at 200 μg/ml refolding buffer resulted in only 4.7% of native protein.     

In vitro and in vivo survival of frozen-thawed bovine oocytes after IVF,nuclear transfer,and parthenogenetic activation

Cryopreservation of bovine oocytes would be beneficial both for nuclear transfer and for preservation efforts. The overall objective of this study was to evaluate the viability as well as the cryodamage to the nucleus vs. cytoplasm of bovine oocytes following freezing-thawing of oocytes at immature (GV) and matured (MII) stages using in vitro fertilization (IVF), parthenogenetic activation, or nuclear transfer assays. Oocytes were collected from slaughterhouse ovaries. Oocytes at the GV, MII, or MII but enucleated (MIIe) stages were cryopreserved in 5% (v/v) ethylene glycol; 6% (v/v) 1,2-propanediol; and 0.1-M sucrose in PBS supplemented with 20% (v/v) fetal bovine serum. Frozen-thawed oocytes were subjected to IVF, parthenogenetic activation, or nuclear transfer assays. Significantly fewer GV oocytes survived (i.e., remained morphologically intact during freezing-thawing) than did MII oocytes (47% vs. 84%). Subsequent development of the surviving frozen-thawed GV and MII oocytes was not different (58% and 60% cleavage development; 7% and 12% blastocyst development at Day 9, respectively, P > 0.05). Parthenogenetic activation of frozen-thawed oocytes resulted in significantly lower rates of blastocyst development for the GV than the MII oocyte groups (1% vs. 14%). Nuclear transfer with cytoplasts derived from frozen-thawed GV, MII, MIIe, and fresh-MII control oocytes resulted in 5%, 16%, 14%, and 17% blastocyst development, respectively. However, results of preliminary embryo transfer trials showed that fewer pregnancies were produced from cloned embryos derived from frozen oocytes or cytoplasts (9%, n = 11 embryos) than from fresh ones (19%, n = 21 embryos). Transfer of embryos derived by IVF from cryopreserved GV and MII oocytes also resulted in term development of calves. Our results showed that both GV and MII oocytes could survive freezing and were capable of developing into offspring following IVF or nuclear transfer. However, blastocyst development of frozen-thawed oocytes remains poorer than that of fresh oocytes, and our nuclear transfer assay suggests that this poorer development was likely caused by cryodamage to the oocyte cytoplasm as well as to the nucleus. Mol. Reprod. Dev. 51:281–286, 1998. © 1998 Wiley-Liss, Inc.     

Prostanoid formation in primary astroglial cell cultures: Ca-dependency and stimulation by A 23187, melittin and phospholipases A2 and C

Prostaglandin (PG) and thromboxane B₂ (TXB₂) biosynthesis was studied in cultured astrocytes from neonatal rat brain hemispheres. After two weeks of cultivation, prostanoids were formed with the spectrum: PGD₂ > TXB₂ > PGF₂ > PGE₂, as measured by specific radioimmunoassays. Under basal conditions PGD₂ biosynthesis (9.55 ng/mg protein/15 min) was in the same order of magnitude as the sum of the other prostanoids. The formation of prostanoids was stimulated in a concentration dependent manner (up to 6–10 fold) by the calcium ionophore A 23187 (0.01–10 μM) as well as by melittin (0.01–5 μg/ml), phospholipase A₂ (10–40 U/ml) and phospholipase C (0.01–1 U/ml). Basal and evoked PG and TXB₂ biosynthesis depended on the availability of Ca²⁺, as demonstrated in Ca²⁺ free incubation medium containing Na₂EDTA (1 μM), or with verapamil (100 μM) and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)-octylester-HCl (TMB-8, 1–100 μM). Indomethacin (10 μM), mepacrine (100 μM) and p-bromophenacylbromide (50 μ M) inhibited basal and evoked PG formation. Thin-layer chromatography (TLC) detection after incubation of the cells with [³H]arachidonic acid (1 μCi/ml, for 60 min) confirmed the results obtained by radioimmunoassay. Incubation of [³H]arachidonic acid labelled cells with inonophore or phospholipases, followed by lipid extraction and TLC, showed that A 23187 liberated [³H]arachidonic acid predominantly from phosphatidylethanolamine, whereas phospholipase A₂ and C reduced mainly the labelling of the phosphatidyl-inositol/-choline fraction. Potassium depolarization of the cells did not enhance prostanoid formation. Similarly, drugs with affinity to - or β-adrenoceptors, or to dopamine-, 5-hydroxytryptamine-, muscarine-, histamine-, glutamate-, aspartate-, GABA, adenosine- and opioid-receptors failed to stimulate prostanoid biosynthesis. Also compounds like angiotensin, bradykinin and thrombin were ineffective in this respect.

In conclusion, our results confirm that cultured astrocytes possess the complete pattern of enzymes necessary for prostanoid formation and hence might play a crucial role in brain prostanoid biosynthesis. Stimulation of prostanoid biosynthesis involves Ca²⁺-dependent activation of phospholipase A₂, cyclooxygenase reaction and further PG metabolism. However, the endogenous stimulus for enhanced prostanoid synthesis in the brain still has to be established.     

Differential T cell-mediated regulation of CD23 (Fc epsilonRII) in B cells and follicular dendritic cells

Differences in murine follicular dendritic cells (FDC)-CD23 expression under Th1 vs Th2 conditions prompted the hypothesis that T cells help regulate the phenotype of FDCs. FDCs express CD40, suggesting that T cell-CD40L and lymphokines may be involved in regulating FDC-CD23. To test this, highly enriched FDCs were incubated with CD40L trimer or anti-CD40 to mimic T cell signaling in the presence of IFN-gamma or IL-4. Surface expression of CD23 was determined by flow cytometry, whereas mRNA levels of CD23 and its isoforms CD23a and CD23b were independently measured by quantitative PCR. When FDCs were incubated with either CD40L trimer or agonistic anti-CD40 Ab, the expression of FDC-CD23 was increased both at the mRNA and protein levels. Moreover, engagement of FDC-CD40 enhanced mRNA levels for both CD23a and CD23b isoforms. In addition, IFN-gamma substantially enhanced CD23a and CD23b mRNA levels in CD40-stimulated FDCs. Curiously, IL-4 could also up-regulate FDC-CD23a but not -CD23b. Anti-IFN-gamma dramatically inhibited FDC-CD23 in mice immunized with CFA, whereas anti-IL-4 had only a modest inhibitory effect. In contrast with FDCs, IFN-gamma inhibited surface expression of murine B cell-CD23 as well as mRNA for B cell CD23a and -CD23b, whereas IL-4 dramatically enhanced message for both isoforms as well as protein expression. In short, CD23 was regulated very differently in FDCs and B cells. Previous studies suggest that high levels of FDC-CD23 inhibit IgE production, and this IFN-gamma and CD40L-mediated up-regulation of FDC-CD23 may explain, at least in part, why Th1 responses are associated with low IgE responses in vivo.     

Predicting disulfide bond connectivity in proteins by correlated mutations analysis

MOTIVATION: Prediction of disulfide bond connectivity facilitates structural and functional annotation of proteins. Previous studies suggest that cysteines of a disulfide bond mutate in a correlated manner. RESULTS: We developed a method that analyzes correlated mutation patterns in multiple sequence alignments in order to predict disulfide bond connectivity. Proteins with known experimental structures and varying numbers of disulfide bonds, and that spanned various evolutionary distances, were aligned. We observed frequent variation of disulfide bond connectivity within members of the same protein families, and it was also observed that in 99% of the cases, cysteine pairs forming non-conserved disulfide bonds mutated in concert. Our data support the notion that substitution of a cysteine in a disulfide bond prompts the substitution of its cysteine partner and that oxidized cysteines appear in pairs. The method we developed predicts disulfide bond connectivity patterns with accuracies of 73, 69 and 61% for proteins with two, three and four disulfide bonds, respectively.